The present invention relates to a method of detection for human Spinocerebellar ataxia 2 gene variants, and more particularly their use in applications such as molecular diagnosis, prediction of an individual""s disease susceptibility, and the genetic analysis of SCA2 gene in a population. The invention also provides primer and probe sequences useful in detecting these polymorphic variations in SCA2 gene and their use in diagnosis and prediction of an individual""s susceptibility to SCA2 disease.
Spinocerebellar ataxias (SCAs) are a clinically heterogeneous group of autosomal dominant neurodegenerative disorders characterized by progressive deterioration in balance and coordination. The clinical symptoms include ataxia, dysarthria, ophthalmoparesis, and variable degrees of motor weakness. The symptoms occur due to progressive neuronal loss primarily in the cerebellum but also in other parts of central nervous system. The symptoms usually begin during the third or fourth decade of life, however, juvenile onset has been identified. Typically, the disease worsens gradually, often resulting in complete disability and death 10-20 years after the onset of symptoms. Individuals with juvenile onset spinocerebellar ataxias, however, typically have more rapid progression of the phenotype than the late onset cases.
Seven disease loci have been identified to date as causing this phenotypexe2x80x94Spinocerebellar ataxia 1 (SCA1) (Orr et al., Nat. Genet. 4, 221-226 (1993)), SCA2 (Pulst et al., Nat. Genet. 14, 269-276 (1996); Sanpei et al., Nat. Genet. 14, 227-284 (1996); Imbert et al., Nat. Genet. 14, 285-291 (1996)), SCA3/MJD (Kawaguchi et al., Nat. Genet. 8, 221-227 (1994)), SCA6 (Zhuchenko et al, Nat. Genet. 15,62-68 (1997)), SCA7 (David et al., Nat. Genet. 17, 65-70 (1997)), SCA8 (Koob et al., Nat. Genet. 21, 379-384 (1999)) and SCA12 (Holmes et al., Nat. Genet. 23, 391-392 (1999)). The causative mutation associated with all these disease types is abnormal expansion of trinucleotide repeat motif in their corresponding gene. The expansion of the repeat tract beyond the normal range produces premutation allele that may further expand to disease producing mutations.
The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution generating variant forms of progenitor sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). The variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species, is eventually incorporated into the DNA of many or most members of the species, and effectively becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms. Several different types of polymorphisms have been reported. A restriction fragment length polymorphism (RFLP) means a variation in DNA sequence that alters the length of a restriction fragment as described in Botstein et al., Am. J. Hum. Genet. 32, 314-331 (1980). The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses (Donis-Keller, Cell 51, 319-337 (1987)). Other polymorphisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetranucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analysis and in a large number of genetic mapping studies.
Other polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPS, STRs and VNTRs. Some single nucleotide polymorphisms (SNPs) occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Examples of genes, in which polymorphisms within coding sequences give rise to genetic disease include beta.-globin (sickle cell anemia) and CFTR (cystic fibrosis). Other single nucleotide polymorphisms occur in non-coding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymorphisms have no phenotypic effects.
SNPs can be used in the same manner as RFLPs, and VNTRs but offer several advantages. SNPs occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. The greater frequency and uniformity of SNPs means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms. Also, the different forms of characterized SNPs are often easier to distinguish that other types of polymorphism (e.g., by use of assays employing allele-specific hybridization probes or primers).
Spinocerebellar ataxia 2 (SCA2), which was initially described in a Cuban population (Gispert et al., Nat. Genet. 4, 295-299 (1993)), has now been reported worldwide. The human SCA2 gene has 25 exons and encompasses approximately 130 kb on 12q23-24.1 region of chromosome 12 (Sahba et al., Genomics 47, 359-364 (1998)). The molecular basis of the disease is an expansion of a CAG repeat tract in exon 1 of SCA2 gene. The molecular diagnosis of clinically suspected SCA2 patients is carried out by the correct sizing of the CAG repeats at the SCA2 locus. In normal individuals this CAG repeat is not only polymorphic in length, ranging from 14-31 repeats with a mode of 22 repeats, but also cryptic in nature, having one or more interrupting CAA triplets. In contrast, the SCA2 disease alleles contain a pure, contiguous stretch of 34-59 CAG repeats. Sanpei and Tsuji (patent CA2241173, EP00878543 and WO 98/18920) have provided the cDNA fragments of the gene causative of spinocerebellar ataxia type 2 having a determined base sequence. Pulst and Ramos in patent WO 97/42314 have also provided the isolated nucleic acids encoding human SCA2 protein or fragments thereof and a method of diagnosis of SCA2 disease.
Tsuji and Sanpei have also patented a method for specifically diagnosing SCA2 (patents CA22323 11, EP0869186 and WO 98/03679). Therein the method comprises effecting PCR by employing DNA to be tested as template and using nucleic acid primers hybridizable with the parts of the base sequences of the SCA2 gene. The diagnosis depends on the number of the CAG repeat units in the SCA2 gene, the patient with SCA2 has the number of CAG repeat units of 35 or above while the gene of a normal subject has 15 to 24 repeats, which enables the diagnosis of SCA2.
However, these methods are not useful for detecting normal individuals carrying repeats predisposed to instability and expansion (premutation alleles) as the repeat length alone would not be the correct predictor of repeat instability at SCA2 locus due to presence of varying number of CAA interruptions. The presence of interruptions within the triplet repeats has been shown to play an important role in determining stability to a number of trinucleotide repeat disorders (Chung et al., Nat. Genet. 5, 254-258 (1993); Kunst et al., Cell 77, 853-861 (1994); Eichler et al., Nat. Genet. 8, 88-94 (1994)). It has been proposed that the presence of these interruptions confers stability and their absence predisposes alleles to instability and eventual disease status.
The prior art is lacking in any method that associates the allelic variants of SCA2 gene to the disease susceptibility. The prior art is also lacking in any study that correlates the substructure of SCA2 CAG repeat with repeat instability and predisposition to the SCA2 disease. This is the first demonstration that relates to the detection of single nucleotide polymorphisms in human SCA2 gene and their use for applications such as molecular diagnosis, prediction of an individual""s SCA2 disease susceptibility or otherwise, and/or the genetic analysis of SCA2 gene in a population. The novelty of present invention is in providing a method for detecting allelic variants of SCA2 gene within the human population and their association with the disease for prediction of an individual""s predisposition to SCA2.
The main object of the present invention is to provide method of detection of allelic variants of human SCA2 gene.
Another object is to provide allele specific primers and probes useful for detection of allelic variants of human SCA2 gene.
Yet another object of the invention is to provide a method for establishing association of SCA2 allelic variants with disease susceptibility.
Still another object of the invention is to provide a method for screening individuals carrying SCA2 alleles predisposed to instability and expansion.
The present invention relates to allelic variants of human Spinocerebellar ataxia 2 (SCA2) gene and provides allele-specific primers and probes suitable for detecting these allelic variants for applications such as molecular diagnosis, prediction of an individual""s disease susceptibility, and/or the genetic analysis of SCA2 gene in a population.
The present invention relates to the detection of the allelic variants of the human SCA2 gene and their utility in predicting an individual""s susceptibility to the SCA2 disease.
Accordingly, the present invention provides method detection of human Spinocerebellar ataxia 2 gene variants, said method comprising the steps of:
1. designing and synthesizing oligonucleotide primers for PCR amplification of CAG repeat containing region of exon 1 of human SCA2 gene,
2. amplifying genomic DNA of SCA2 patients and normal control individuals using the above said primers,
3. sequencing the amplified PCR product and identifying sequence variations computationally by comparing it with the already existing sequence of human SCA2 gene,
4. screening normal control individuals and SCA2 patients for novel single nucleotide polymorphisms using allele specific oligonucleotide probes,
5. computing the frequencies of CC and GT haplotypes in normals and SCA2 patients,
6. establishing the association of the CC and GT haplotype with the SCA2 disease based on their frequency distribution in normals and SCA2 patients,
7. predicting the risk or susceptibility to the SCA 2 disease based on the haplotype present at the polymorphic sites in the individual tested, GT haplotype being at low risk and CC haplotype at high risk to the disease.
In an embodiment, the primers suitable for amplification of the SCA2 gene region containing one or more polymorphic sites, are selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and compliments thereof.
In another embodiment, the allele specific oligonucleotide probes useful for detection of SCA2 gene variants are selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and the compliments thereof, wherein the polymorphic site occupies a central position of the probe.
In yet another embodiment, the length of the oligonucleotide primers and probes is in the range of 5 to 100 bases.
In still another embodiment, allelic variants of SCA2 gene have GT and CC haplotypes, Further, the invention provides a diagnostic kit for the detection of SNP haplotypes (CC/GT) comprising suitable primers and probes selected from polynucleotide sequences under SEQ ID NO: 1 to 12.
In another embodiment of the invention a nucleic acid vector may contain the allelic variants of SCA2 gene.
In an embodiment of the invention primers suitable for amplification of SCA2 gene region containing one or more polymorphic sites are provided, said primers selected from the group comprising:
a) CTC CGC CTC AGA CTG TTT TGG TAG 3xe2x80x2 (as listed in SEQ ID NO: 1); and
b) GTG GCC GAG GAC GAG GAG AC 3xe2x80x2 (as listed in SEQ ID NO: 2) and compliments thereof.
In yet another embodiment of the invention allele specific primers suitable for detection of allelic variants of SCA2 gene are provided, selected from the group comprising:
a) 5xe2x80x2 CTC GGC GGG CCT CCC CGC CCC TTC GTC GTC C 3xe2x80x2 (as listed in SEQ ID NO: 3);
b) 5xe2x80x2 CTC GGC GGG CCT CCC CGC CCC TTC GTC GTC G 3xe2x80x2 (as listed in SEQ ID NO: 4);
c) 5xe2x80x2 CCT CCC CGC CCC TTC GTC GTC 3xe2x80x2 (as listed in SEQ ID NO: 5);
d) 5xe2x80x2 CGC CAA CCC GCG CCT CCC CGC TCG GCG CCC GC 3xe2x80x2 (as listed in SEQ ID NO: 6);
e) 5xe2x80x2 CGC CAA CCC GCG CCT CCC CGC TCG GCG CCC GT 3xe2x80x2 (as listed in SEQ ID NO: 7); and
f) 5xe2x80x2 GCG CCT CCC CGC TCG GCG CCC G 3xe2x80x2 (as listed in SEQ ID NO: 8) and compliments thereof.
In still another embodiment of the invention allele specific probes useful for detection of SCA2 gene variants wherein the polymorphic site occupies a central position of the probe are provided, said allele specific probes selected from the group comprising:
a) 5xe2x80x2 CCC CTT CGT CGT CCT CCT TCT CCC CCT 3xe2x80x2 (as listed in SEQ ID NO: 9);
b) 5xe2x80x2 CCC CTT CGT CGT CGT CCT TCT CCC CCT 3xe2x80x2 (as listed in SEQ ID NO: 10);
c) 5xe2x80x2 CGC TCG GCG CCC GCG CGT CCC CGC CGC 3xe2x80x2 (as listed in SEQ ID NO: 11); and
d) 5xe2x80x2 CGC TCG GCG CCC GTG CGT CCC CGC CGC 3xe2x80x2 (as listed in SEQ ID NO: 12) are compliments thereof.
The allelic variants of human SCA2 gene may comprise one or more of the following single nucleotide polymorphisms as compared with the human SCA2 complete cDNA sequence in the data base (GenBank accession number U70323).
The sites of change is in accordance with the human SCA2 complete cDNA sequence in the database (GenBank accession number U70323).
The invention also provides a method of analysing a nucleic acid from an individual for the presence of base at any one of the polymorphic sites shown in Table 1. This type of analysis can be performed on a plurality of individuals who are tested either for the presence or for the predisposition to the SCA2 disease. The susceptibility to the disease can then be established based depending on the base or set of bases present at the polymorphic sites in the individuals tested.
The invention also provides oligonucleotide sequences (as listed in SEQ ID NO: 1 to 12), suitable for use as allele specific primers and probes for the detection of polymorphic sites listed in Table 1.
Further, a diagnostic kit comprising one or more of the allele specific primers or probes along with the required buffers and accessories suitable for identification of SCA2 allelic variants to establish an individual""s susceptibility to SCA2 disease is also included in the invention.
Eucaryotic expressing vectors comprising a DNA sequence coding for a protein or a peptide according to the invention are new materials and are also included in the invention. Host cells, for example, cloned human cell lines, can be transformed using the new expression vectors and are also included in the invention.